The long-term goal of this research is to understand the molecular mechanisms that regulate gene expression during development. Our model system has been the Drosophila alcohol dehydrogenase (Adh) gene, a single-copy gene that is transcribed from two different promoters, the distal (adult) and the proximal (embryonic-larval), in different tissues and at different times during development. Our specific aims are to investigate the mechanisms by which chromatin structure, cis-acting sequences, trans-acting factors and their interactions affect the regulation of the Adh gene. Specific aspects of the Adh gene regulation are found in Drosophila tissue culture cells. Probing chromatin of ADH- and ADH+ cells with nucleases has shown that localized, defined chromatin structural changes accompany specific transcription from the distal promoter. In situ Exonuclease III protection assay of ADH- and ADH+ chromatin has demonstrated differences in DNA-protein interactions in vivo at specific 5' regions of Adh. These sites of specific DNA-protein interactions will be analyzed further at single-nucleotide resolution in vivo by genomic footprinting in nuclei and intact cells. The functional significance of the specific protein-binding DNA sequences will be determined by generating defined, small deletions and point mutations. The functional assays of these mutants will be the in vivo transient expression in Drosophila tissue culture cells of different types: ADH- and ADH+ cells that provide different trans-acting factors. Nuclear extracts prepared from ADH- and ADH+ cell lines have been shown to contain cell-type specific, protein factors that bind to DNA 5' of the distal RNA start site. These sequence-specific DNA-binding factors, which includes a novel factor whose specific binding activity appears abundant in ADH- than ADH+ cells, will be examined in more cell lines and tissues from different developmental stages. Biochemical isolation of these factors will be attempted by uses of sequence- specific DNA affinity chromatography. The knowledge obtained from this study should increase our understanding of how gene activity is correctly timed and localized during growth of complex organisms.